Herpesviruses are a group of ubiquitous viruses which bear both structural and compositional resemblance. Most agents of the herpesvirus group can integrate their DNA into that of the host cell (particularly lymphoid cells or ganglia) after infecting the human host, thereby causing a latent infection which has the potential to reactivate and cause recurrent disease. There are at least six herpesviruses which are known to frequently infect humans, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and Human herpesvirus 6 (HHV-6).
2.1 Herpes Simplex Virus Infection
Herpes simplex virus (HSV) causes, or is associated with, a wide variety of diseases in humans (Table 1).
TABLE 1 ______________________________________ Diseases caused by Herpes Simplex Virus ______________________________________ genital lesions stomatitis encephalitis herpetic dermatitis meningitis pharyngitis keratoconjunctivitis pneumonia neonatal herpes keratitis chorioretinitis herpetic hepatitis eczema herpeticum erythema multiforme ______________________________________
There are instances in which rapid, sensitive, and specific diagnosis of HSV disease is imperative. The most serious HSV infection is encephalitis. Encephalitis is often a disseminated infection in newborns which may be acquired either during or after birth; and an adult infection affecting the temporal lobe of the brain. Currently, definitive diagnosis is by brian biopsy and culture to isolate HSV. Serologic diagnosis, particularly of HSV in cerebrospinal fluid (CSF), is not sufficiently sensitive or specific, and takes too much time to be of use in decisions involving choices for early therapeutic intervention of encephalitis. Early therapy of patients with encephalitis, before irreversible hemorrhagic necrosis of the brain, has resulted in improved outcomes.
Further, due to the high morbidity and mortality of infants having neonatal herpes infection, and since many cases of neonatal HSV infection can be prevented by cesarean section, diagnosis of maternal infection before delivery is important. Since cultures of the mother taken days or weeks before delivery do not predict well whether the mother may be symptomatic at the time of delivery, a rapid, sensitive, and specific assay for detecting HSV in body fluids or secretions is desirable as a means to monitor infection, and consequently, determine the necessity of cesarean section.
Additionally, there are instances in which knowing the infecting serotype is helpful; i.e. whether infection is cause by HSV-1 or HSV-2. Since both HSV-1 and HSV-2 share antigens, serological differentiation is difficult. Distinguishing between infection caused by HSV-1 and HSV-2 may be important because it has been reported that sensitivity to antiviral therapy can vary with the serotype. Identifying the serotype can also provide prognostic information. For example, genital infections caused by HSV-1 are less likely to recur than those infections caused by HSV-2.
2.2 Diagnosis of HSV Infection
Current methods for the detection of HSV in clinical specimens have been routinely accomplished by using the clinical specimens to infect susceptible cell lines in amplifying the amount of virus (antigen) to increase the sensitivity and specificity of detection; isolating the virus from the cultured cell lines; and subsequently immunologically confirming the virus identity. The disadvantages of such methods of detection include that the time required for complete diagnosis can range from 1-12 days, depending on the sensitivity of the cells inoculated and the amount of virus in the inoculum; and that the methods are both labor and time intensive, and expensive.
Immunological assays, including immunofluorescence (IF) or enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (RIA) usually have good sensitivity. However, such immunoassays can suffer from either lack of good specificity or sensitivity, and lack of good reproducibility. For example, certain clinical specimens, such as vesicle fluid and mucus or other body secretions, can bind nonspecifically to proteins such as antibodies or antigen. Sensitivity depends on the titer of antibody found in the clinical specimen, which in the case of herpes simplex encephalitis, occurs in the CSF weeks after infection. Additionally, early therapeutic intervention (e.g., with acyclovir) can attenuate antibody response after some primary infections (Kahlon et al., 1987, J. Infect. Dis. 155:38-43). Reproducibility can vary with the lot of antisera used and/or the lot of microtitration plates, which may vary in their binding abilities.
Recent advances in molecular biology have spurred the use of DNA probes in attempts to provide a more rapid, sensitive and specific assay for detecting HSV in clinical specimens. For example, a radiolabeled DNA probe has been used to hybridize to tissue cultures infected with or by HSV, or in clinical samples suspected of containing HSV ("hybridization assays"). However, probing of tissue cultures requires at least 18-24 hours for growth to amplify the antigen (HSV) to be detected, if present, and further time for development of autoradiographic detection systems. Using hybridization assays for assaying clinical specimens for HSV may lack sensitivity, depending upon the titer of virus and the clinical sample assayed. Detection of HSV in clinical samples has been reported using the polymerase chain reaction (PCR) to enzymatically amplify HSV DNA (Cao et al., 1989, J. Invest. Dermatol. 92:391-392; Powell et al., 1990, Lancet, 335:357-358; Rowley et al., 1990, Lancet, 335:440-441; and Aurelius et al., 1991, Lancet, 337:189-192). However, because of the dangers of false positive reactions, these procedures require rigid controls to prevent contamination and carry over (Ehrlich et al., 1994, pp.3-18 in PCR-Based Diagnostics in Infectious Diseases, GD Ehrlich and SJ Greenberg (eds), Blackwell Scientific Publications). Therefore, there exists a need for a rapid, sensitive, and specific assay for herpesvirus, HSV-1 and HSV-2.
2.3 Cytomegalovirus Infection
Cytomegalovirus (CMV) causes, or is associated with, a wide variety of diseases in humans (Table 2). More than 90% of bone marrow or kidney transplant recipients (immunocompromised hosts) develop CMV infections, most of which are due to reactivation of latent virus by immunosuppressive drugs, as well as transmission of virus by latently infected donor tissue or blood (Ackerman et al., 1988, Transplant. Proc. 20(S1):468-71; Peterson et al., 1980, Medicine 59:283-300).
TABLE 2 ______________________________________ Diseases caused by Cytomegalovirus ______________________________________ cytomegalic inclusion heterophil-negative disease in neonates mononucleosis interstitial pneumonia pneumonitis retinitis hepatitis pancreatitis meningoencephalitis gastrointestinal disease disseminated infection ______________________________________
2.4 Diagnosis of CMV Infection
There are instances in which rapid, sensitive, and specific diagnosis of CMV disease is imperative. In recent years, the number of patients undergoing organ and tissue transplantations has increased markedly. CMV is the most frequent cause of death in immunocompromised transplant recipients, thereby confirming the need for rapid and reliable laboratory diagnosis. Lymphocytes, monocytes, and possibly arterial endothelial or smooth muscle cells, are sites of CMV latency. Therefore, prevention of CMV infections in immunocompromised individuals (e.g., transplant recipients) includes use of CMV-negative blood products and organs. Additionally, CMV can be spread transplacentally, and to newborns by contact with infected cervical secretions during birth. Thus, a rapid, sensitive, and specific assay for detecting CMV in body fluids or secretions may be desirable as a means to monitor infection, and consequently, determine the necessity of cesarean section.
Diagnosis of CMV infection may be performed by conventional cell culture using human fibroblasts; shell vial centrifugation culture utilizing monoclonal antibodies and immunofluorescent staining techniques; serological methods; the CMV antigenemia assay which employs a monoclonal antibody to detect CMV antigen in peripheral blood leukocytes (PBLs); or by nucleic acid hybridization assays. These various methods have their advantages and limitations. Conventional cell culture is sensitive but slow, as cytopathic effect (CPE) may take 30 or more days to develop. Shell vial centrifugation is more rapid but still requires 24-48 hours for initial results. Both culture methods are affected by antiviral therapy. In immunocompromised patients, the ability to mount IgG and/or IgM antibody responses to CMV infection are impaired, and serological methods are thus not reliable in this setting. Alternatively, IgM antibodies may be persistent for months after infection is resolved, and thus their presence may not be indicative of active infection. The CMV antigenemia assay is labor intensive and is not applicable to specimens other than PBLs.
Recent advances in molecular biology have spurred the use of DNA probes in attempts to provide a more rapid, sensitive and specific assay for detecting CMV in clinical specimens. For example, radiolabeled DNA probes have been used to hybridize to tissue cultures infected with or by CMV, or in clinical samples suspected of containing CMV ("hybridization assays"). However, probing of tissue cultures requires at least 18-24 hours for growth to amplify the antigen (CMV) to be detected, if present, and additional time for development of autoradiographic detection systems. Using hybridization assays for assaying clinical specimens for CMV may lack sensitivity, depending upon the titer of virus and the clinical sample assayed. Detection of CMV in clinical samples has been reported using the polymerase chain reaction (PCR) to enzymatically amplify CMV DNA. Methods using PCR compare favorably with virus isolation, in situ hybridization assays, and Southern blotting (See for example, Bamborschke et al., 1992, J. Neurol. 239:205-208; Drouet et al., 1993, J. Virol. Methods 45:259-276; Einsele et al., 1991, Blood 77:1104-1110; Einsele et al., 1991, Lancet 338:1170-1172; Lee et al., 1992, Aust. NZ J. Med. 22:249-255; Miller et al., 1994, J. Clin. Microbiol. 32:5-10; Rowley et al., 1991, Transplant. 51:1028-1033; Spector et al., 1992, J. Clin. Microbiol. 30:2359-2365; and Stanier et al., 1992, Molec. Cell. Probes 8:51-58). Others, comparing the CMV antigenemia assay with PCR methods, have found PCR methods as efficient or slightly more efficient in the detection of CMV (van Dorp et al., 1992, Transplant. 54:661-664; Gerna et al., 1991, J. Infect. Dis. 164:488-498; Vleiger et al., 1992, Bone Marrow Transplant. 9:247-253; Zipeto et al., 1992, J. Clin. Microbiol. 30:527-530). In addition, PCR methods have exhibited great sensitivity when specimens other than PBLs are assayed (Natori et al., 1993, Kansenshogaku Zasshi 67:1011-1015; Peterson et al., 1980, Medicine, 59:283-300; Prosch et al., 1992, J. Med. Virol. 38:246-251; Ratnamohan et al., 1992, J. Med. Virol. 38:252-259). However, because of the dangers of false positive reactions, these procedures require rigid controls to prevent contamination and carry over (Ehrlich et al., 1994, pp.3-18 in PCR-Based Diagnostics in Infectious Diseases, GD Ehrlich and SJ Greenberg (eds), Blackwell Scientific Publications). Therefore, there exists a need for a rapid, sensitive, and specific assay for herpesvirus, CMV.